Prosaposin-derived peptides

ABSTRACT

The invention provides a method of alleviating neuropathic pain in a subject by administering an effective amount of an active fragment of prosaposin to the subject. The invention also provides a method of preventing neuropathic pain in a subject by administering an effective amount of an active fragment of prosaposin to the subject.

This is a continuation of U.S. application Ser. No. 08/611,307, filedMar. 5, 1996, (pending), the disclosure of which is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of pain therapy and morespecifically to the use of prosaposin-derived peptides for the treatmentof neuropathic pain.

2. Background Information

Neuropathic pain results from injury to a nerve. In contrast to theimmediate pain caused by tissue injury, neuropathic pain can developdays or months after a traumatic injury. Furthermore, while pain causedby tissue injury is usually limited in duration to the period of tissuerepair, neuropathic pain frequently is long-lasting or chronic.Moreover, neuropathic pain can occur spontaneously or as a result ofstimulation that normally is not painful.

The clinical causes of neuropathic pain are widespread and include bothtrauma and disease. For example, traumatic nerve compression or crushand traumatic injury to the brain or spinal cord are common causes ofneuropathic pain. Furthermore, most traumatic nerve injuries also causethe formation of neuromas, in which pain occurs as a result of aberrantnerve regenration. In addition, cancer-related neuropathic pain iscaused when tumor growth painfully compresses adjacent nerves, brain orspinal cord. Neuropathic pain also is associated with diseases such asdiabetes or alcoholism.

Unfortunately, neuropathic pain frequently is resistant to availabledrug therapies. In addition, current therapies have serious side-effectsincluding, for example, cognitive changes, sedation, nausea and, in thecase of narcotic drugs, addiction. Many patients suffering fromneuropathic pain are elderly or have other medical conditions thatparticularly limit their tolerance of the side-effects associated withavailable drug therapy. The inadequacy of current therapy in relievingneuropathic pain without producing intolerable side-effects frequentlyis manifest in the depression and suicidal tendency of chronic painsufferers.

Methods of alleviating neuropathic pain would improve the quality oflife for many people suffering from pain due to trauma or disease.However, there currently are not effective drugs that relieveneuropathic pain without undesirable side-effects such as sedation andaddiction. Thus, there is a need for methods of alleviating neuropathicpain without producing undesirable side-effects. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of alleviating neuropathic painin a subject by administering an effective amount of an active fragmentof prosaposin to the subject. For example, the invention provides amethod of alleviating neuropathic pain resulting from a disorder ofperipheral nerve, dorsal root ganglia, spinal cord, brain stem, thalamusor cortex in a subject by administering an effective amount of an activefragment of prosaposin having the amino acid sequenceCys—Flu—Phe—Leu—Val—Lys—Glu—Val—Thr—Lys—Leu—Ile—Asp—Asn—Asn—Lys—Thr—Glu—Lys—Glu—Ile—Leu(SEQ ID NO: 1) orThr—D—Ala—Leu—Ile—Asp—Asn—Asn—Ala—Thr—Glu—Glu—Ile—Leu—Tyr (SEQ ID NO:2). In addition, the invention provides a method of preventingneuropathic pain in a subject by administering an effective amount of anactive fragment of prosaposin to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the threshold of tactile allodynia before (time 0) and atvarious times after bolus injection of prosaposin-derived 22-mer peptide(SEQ ID NO: 1) in Chung model rats.

FIG. 2 show the threshold of tactile allodynia before (time 0) and atvarious times after bolus injection of prosaposin-derived 14-mer peptide(SEQ ID NO: 2) in Chung model rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of alleviating neuropathic painin a subject by administering an effective amount of an active fragmentof prosaposin to the subject. As disclosed herein, the method of theinvention can alleviate neuropathic pain in a subject within 30 minutesof administration. Such a method is useful for alleviating neuropathicpain resulting from a disorder of peripheral nerve, dorsal root ganglia,spinal cord, brainstem, thalamus or cortex.

A peptide useful in the invention is derived from prosaposin, which is a517 amino acide protein originally identified as the precursor of foursphingolipid activator proteins (Kishimoto et al., J. Lipid Res.,33:1255-1267 (1992)). Four adjacent tandem domains in prosaposin areproteolytically processed in lysosomes to generate saposins A, B, C, andD, which activate hydrolysis of glycospingolipids by lysosomalhydrolases (O'Brien and Kishimoto, FASEB J., 5:301-308 (1991)).

The unprocessed form of prosaposin is found in high concentrations inhuman and rat brain, where it is localized within neuronal surfacemembranes. During embryonic development, prosaposin mRNA is abundant inbrain and dorsal root ganglia. Furthermore, prosaposin binds with highaffinity to gangliosides, which stimulate neurite outgrowth, andpromotes transfer of gangliosides from micelles to membranes.

The neurotrophic activity of prosaposin is consistent with itslocalization in neuronal cell populations (O'Brien et al., Proc. Natl.Acad. Sci., USA 91:9593-9596 (1994); San et al., Biochem. Biophys. Res.Commun., 204:994-1000 (1994), each of which is incorporated herein byreference). Prosaposin stimulates motor neurite outgrowth in vitro andin vivo and increases choline acetyltransferase activity, which is amarker of neuronal differentiation. In addition, prosaposin preventscell death in neuroblastoma cells (O'Brien et al., supra, 1994; O'Brienet al., FASEB J. 9:681-685 (1995), which is incorporated herein byreference).

The enurotrophic activity of prosaposin is localized to saposin C, adomain of 80 amino acids. A 22-mer peptide corresponding to amino acids8 to 29 of the saposin C domain (SEQ ID NO: 1) stimulates neuriteoutgrowth and choline acetyltransferase activity and prevents cell deathin neuroblastoma cells (O'Brien et al., supra, 1995).

Prosaposin or the prosaposin-derived 22-mer peptide (SEQ ID NO: 1), forexample, can modulate motor neuron function by promoting neuriteoutgrowth. Prior to the present invention, however, it was not knownwhether prosaposin or a peptide fragment of prosaposin could affectsensory neuron function. Moreover, the neurotrophic activity ofprosaposin or a prosaposin-derived peptide in stimulating motor neuriteoutgrowth is evident only after a period of 24 to 48 hours (see, forexample, O'Brien et al., supra, 1994). Neurotrophic activity ofprosaposin or a prosaposin-derived peptide has not been demonstrated tooccur in a shorter period of time.

In contrast, the present invention provides a method of alleviatingneuropathic pain, which involves both sensory and motor neuroncomponents. Furthermore, the method of the invention is effective inalleviating neuropathic pain in a matter of minutes rather than thehours or days previously demonstrated to be required for theneurotrophic activity of prosaposin or a prosaposin-derived peptide.

The effectiveness of the method of the invention in alleviatingneuropathic pain was demonstrated using the well-recognized Chung ratmodel of peripheral neuropathy. In the Chung rat model, spinal nervepartial ligation of left spinal nerves L-5 and L-6 produces along-lasting hypersensitivity to light pressure on the affected leftfoot. The hypersensitivity is similar to the pain experienced by humanswith the neuropathic condition of causlgia (Kim and Chung, Pain50:355-363 (1992), which is incorporated herein by reference).

Prior to administration of an active fragment of prosaposin, Chung modelrats had a threshold of 3.0 to 4.0 g before the affected foot waswithdrawn in response to pressure (Von Frey hairs) (see FIG. 1). Afteradministration of an active fragment of prosaposin (prosaposin-derived22-mer; SEQ. ID NO: 1), neuropathic pain was alleviated, as evidenced bya greater tolerance to pressure before the affected foot was withdrawn.The effect of the active fragment of prosaposin occurred within 15minutes and was sustained for 3 hours following administration as shownin FIG. 1. This rapid relief of neuropathic pain is in stark contrast tothe delayed neurotrophic effects previously reported for prosaposin andpeptides derived from prosaposin.

As used herein, the term “neuropathic pain” means pain resulting frominjury to a nerve. Neuropathic pain is distinguished from nociceptivepain, which is the pain caused by acute tissue injury involving smallcataneous nerves or small nerves in muscle or connective tissue. Paininvolving a nociceptive mechanism usually is limited in duration to theperiod of tissue repair and generally is alleviated by availableanalgesic agents or opioids (Myers, Regional Anesthesia 20:173-184(1995), which is incopriated herein by reference).

Neuropathic pain typically is long-lasting or chronic and often developsdays or months following an initial acute tissue injury. Neuropathicpain can involve persistent, spontaneous pain as well as allodynia,which is a painful response to a stimulus that normal is not painful.Neuropathic pain also can be characterized by hyperalgesia, in whichthere is an accentuated response to a painful stimulus that usually istrivial, such as a pin prick. Unlike nociceptive pain, neuropathic paingenerally is resistant to opioid therapy (Myers, supra, 1995).

The method of the invention is useful in alleviating neuropathic painresulting from a disorder of peripheral nerve, dorsal root ganglia,spinal cord, brainstem, thalamus or cortex. As used herein, the term“disorder” means any trauma, injury, disease or condition resulting inneuropathic pain.

The method of the invention is useful in alleviating neuropathic painregardless of the etiology of the pain. For example, a method of theinvention can be used to alleviate neuropathic pain resulting from aperipehral nerve disorder such as neuroma; nerve compression; nervecrush, nerve stretch or incomplete nerve transaction; mononeuropathy orpolyneuropahty. A method of the invention also can be used to alleviateneuropathic pain resulting from a disorder such as dorsal root ganglioncompression; inflammation of the spinal cord; contusion, tumor orhemisection of the spinal cord; tumors of the brianstem, thalamus orcortex; or trauma to the brainstem, thalamus or cortex (see, forexample, Table 1).

The method of the invention can be useful, for example, to alleviateneuropathic pain resulting from a neuroma, which can develop readilyafter traumatic injury to nerve, especially when a whole nerve isseverely crushed or transsected. In a neuroma, the neurite outgrowththat normally regenerates a peripheral nerve is aberrant or misguideddue, for example, to a physical obstruction such as scar tissue. Thus, aregenerating nerve fiber is entangled in an environment in whichmechanical and physical factors precipitate abnormal eletrophysiologicactivity and pain (Myers, supra, 1995). As amputation neuroma, forexample, can cause phantom pain or can cause pain triggered by the useof a limb prosthesis. As disclosed herein, such neuropathic pain can bealleviated by administration of an active fragment of prosaposinaccording to a method of the invention.

Nerve compression also results in neuropathic pain that can be treatedusing the method of the invention. Nerve compression can be abrupt, asin the cause of traumatic nerve crush, or can be prolonged and moderate,secondary to tumor growth or scar formation in the proximity of a majornerve bundle. Compression neuropathy can occur as a result of changes inblood flow to a nerve, causing severe ischemia and consequent nerveinjury (Myers, supra, 1995).

Administration of an active fragment of prosaposin according to themethod of the invention also can alleviate neuropathic pain resultingfrom a mononeuropathy or polyneuropathy. As used herein, a neuropathy isa functional disturbance or pathological changes in the peripheralnervous system and is characterized clinically by sensory or motorneuron abnormalities. The term mononeuropathy indicates that a singleperipheral nerve is affected, while the term polyneuropathy indicatesthat several peripheral nerves are affected.

TABLE 1 Nerve Neuroma (amputation, nerve transsection) Nerve compression(entrapment neuropathies, tumors) Nerve crush, stretch or incompletetranssection (trauma) Mononeuropathy Diabetes mellitus IrradiationIschemia Vasculitis Polyneuropathy Post-polio syndrome Diabetes mellitusAlcohol Amyloid Toxic HIV Hypothyroidism Uremia Vitamin deficienciesChemotherapy (vincristine, cisplatinum, paclitaxel) ddC (zalcitabine)Fabry's disease Dorsal root ganglion Compression (disk, tumor, scartissue) Root avulsion Inflammation (postherpetic neuralgia) Spinal cordContusion Tumor Hemisection Brainstem, thalamus, cortex Infarction,tumors, trauma

The etiology of a neuropathy can be known or unknown (see, for example,Myers, supra, 1995; Galer, Neurology 45(suppl 9):S17-S25 (1995); Stevensand Lowe, Pathology, Times Mirror international Publishers Limited,London (1995), each of which is incorporated herein by reference). Knownetiologies include complications of a disease or toxic state; forexample, diabetes is the most common metabolic disorder causingneuropathy. The method of the invention alleviates the neuropathic painof a mononeuropathy resulting, for example, from diabetes, irradiation,ischemia or vasculitis. The method of the invention also alleviates theneuropathic pain of a polyneuropathy resulting, for example, frompost-polio syndrome, diabetes, alcohol, amyloid, toxins, HIV,hypothyroidism, uremia, vitamin deficiencies, chemotherapy, ddC orFabry's disease (see Table 1). The method of the invention particularlyis useful in alleviating post-polio myalgia. The method of the inventionalso can alleviate neuropathic pain of unknown etiology.

As disclosed herein, an active fragment of prosaposin can alleviateneuropathic pain. The term “active fragment of prosaposin,” as usedherein, means a peptide that has an amino acid sequence corresponding toan amino acid sequence of prosaposin and that has activity inalleviating neuropathic pain. As used herein, alleviating neuropathicpain means reducing the severity of neuropathic pain. In a humansubject, an active fragment of prosaposin reduces the severity ofneuropathic pain such that the subject's suffering is diminished andquality of life is improved. An active fragment of prosaposin also canalleviate neuropathic pain in any one of a number of well-establishedanimal models of neuropathic pain as described further below (also seeBennett, Muscle & Nerve 16:1040-1048 (1993), which is incorporatedherein by reference). As used herein, the term “active fragment ofprosaposin” is synonymous with “prosaposin-derived peptide”.

The active fragment of prosaposin preferably contains the amino acidsequence Leu—Ile—Asp—Asn—Asn—Lys—Thr—Glu—Lys—Glu—Ile—Leu (SEQ ID NO: 3),which corresponds to amino acids 18 to 29 of saposin C. More preferably,an active fragment of prosaposin has the the amino acid sequenceCyc—Glu—Phe—Leu—Val—Lys—Glu—Val—Thr—Lys—Leu—Ile—Asp—Asn—Asn—Lys—Thr—Glu—Lys—Glu—Ile—Leu(SEQ ID NO: 1), which corresponds to amino acids 8 to 29 of saposin C,or the maono acid sequenceThr—D—Ala—Leu—Ile—Asp—Asn—Asn—Ala—Thr—Glu—Glu—Ile—Leu—Tyr (SEQ ID NO:2), which corresponds to amino acids 16 to 29 of saposin C but which hasbeen modified by a D-alanine for lysine substitution at position 2; analanine for lysine substitution at position 8; a deletion of lysin atposition 11 and the addition of a C-terminal tyrosine residue (see Table2). Such modifications can be useful for increasing peptide stability oruptake across the blood-brain barrier as described below. As usedherein, D-alanine can be represented by D—Ala or X.

An active fragment of prosaposin can have about 12 amino acids to about80 amino acids, which is the full-length of saposin C. Preferably, anactive fragment of prosaposin has about 12 amino acids to about 40 aminoacids and, more preferably, about 14 amino acids to about 22 aminoacids.

TABLE 2 SEQ PEPTIDE SEQUENCE ID NO: Prosaposin-derived 22-merCEFLVKEVTKLIDNNKTEKEIL 1 Prosaposin-derived 14-mer TXLIDNNATEEILY 2Prosaposin-derived 12-mer LIDNNKTEKEIL 3 where X = D-alanine

For use in alleviating neuropathic pain in a human subject, an activefragment of human prosaposin, such as SEQ ID NO: 1 or SEQ ID NO: 2, ispreferred. However, an active fragment derived from another mammalianprosaposin also is useful in alleviating neuropathic pain according tothe method of the invention. Thus, for example, an active fragment ofmouse prosaposin, rat prosaposin, guinea pig prosaposin or bovineprosaposin such as SEQ ID NO: 4 through 7 also can be useful inalleviating neuropathic pain in a subject.

The amino acid sequence of an active fragment of human prosaposin (SEQID NO: 1), which corresponds to amino acids 8 to 29 of saposin C, iswell conserved among other species, as shown in Table 3. In particular,adjacent asparagine (N) residues are conserved among human, mouse, rat,guinea pig and bovine prosaposins. In addition, a leucine (L) residue isconserved 3 to 4 residues toward the N-terminus of the two asparagineresidue and one or more charged residues (aspartic acid (D), lysine (K),glutamic acid (E) or arginine (R)) are conserved 2 to 8 residues towardthe C-terminus of the two asparagine residues. Each of thesewell-conserved residues is underlined in Table 3.

TABLE 3 SPECIES SEQUENCE SEQ ID NO. Human CEFLVKEVTKLIDNNKTEKEIL 1 MouseCQFVMNKFSELIVNNATEELLY 4 Rat CQLVNRKLSELIINNATEELL- 5 Guinea PigCEYVVKKVMLLIDNNRTEEKII 6 Bovine CEFVVKEVAKLIDNNRTEEEIL 7

The well-conserved adjacent asparagine residues, leucine residue andcharged residues described above can be important for the activity of anactive fragment of prosaposin in alleviating neuropathic pain. Forexample, the prosaposin-derived 22-mer (SEQ ID NO: 1) or theprosaposin-derived 14-mer (SEQ ID NO: 2) is an active fragment ofprosaposin, which reduces the painful allodynia seen in the Chung ratmodes of peripheral neuropathy, as disclosed in Example I (see FIGS. 1and 2). In contrast, a mutant 22-mer (SEQ ID NO: 8), which differs fromSEQ ID NO: 1 in having an aspartic acid residue (D) in place of thefirst conserved asparagine (see Table 4), lacks activity in alleviatingneuropathic pain as assayed using Chung rats (see Example I).

The activity of a peptide in alleviating neuropathic pain also cancorrelate with neurotrophic activity. For example, theprosaposin-derived 22-mer (SEQ ID NO: 1) and the prosaposin-derived14-mer (SEQ ID NO: 2) alleviate neuropathic pain and have neurotrophic

TABLE 4 SEQ PEPTIDE SEQUENCE ID NO: Prosaposin-derived 22-merCEFLVKEVTKLIDNNKTEKEIL 1 Mutant 22-mer CEFLVKEVTKLIDDNKTEKEIL 8Prosaposin-derived 14-mer TXLIDNNATEEILY 2 Mutant 14-mer M-1TKLIDNDKTEKEIL 9 Mutant 14-mer M-2 TKSIDNNKTEKEIL 10  where X =D-alanine

activity. In addition, the mutant 22-mer (SEQ ID NO: 8) is inactive inalleviating neuropathic pain as described above and lacks neurotrophicactivity, further indicating that activity in alleviating neuropathicpain can correlate with neurotrophic activity. The mutant 14-mer peptideM-1 (SEQ ID NO: 9), which has a substitution of the second conservedasparagine residue, lacks neurotrophic activity, indicating that peptideSEQ ID NO: 9 also is inactive in alleviating neuropathic pain. Themutant 14-mer peptide M-2 (SEQ ID NO: 10), which has a substitution ofthe conserved leucine residue, lacks neurotrophic activity, indicatingthat peptide SEQ ID NO: 10 is inactive in alleviating neuropathic pain.In contrast, the prosaposin-derived 12-mer peptide (SEQ ID NO: 3), whichhas the conserved-adjacent asparagine, leucine and charged residuesdescribed above, is active as a neurotrophic factor. Thus, theprosaposin-derived 12-mer peptide (SEQ ID NO: 3) also can alleviateneuropathic pain according to the method of the invention.

A peptide useful for alleviating neuropathic pain also can be, forexample, SEQ ID NOS: 11 through 19 (see Table 5). for example, sequencealignment of the prosaposin-derived 22-mer peptide SEQ ID NO: 1 withcytokines and growth factors indicates sequence similarity to a numberof human (h) cytokines including hCNTF, hIL-6, hIL-2, hIL-3, hIL1-γ,erythropoietin (hEPO), human leukocyte inhibitory factor (hLIF), thehIL-1 β chain and oncostatin-M (hONC-M). SEQ ID NOS: 11 through 19, likethe active fragment of prosaposin SEQ ID NO: 1, contain two asparagineresidues that are adjacent or separated by one amino acid. In addition,the cytokine-derived peptide sequences can contain a leucine (L) orisoleucine (I) residue three to four residues toward the N-terminus ofthe two asparagine residues and one or more charged residues (asparticacid (D), lysine (K), glutamic acid (E), or arginine (R)) two to eightresidues toward the C-terminus of the two asparagine residues, as isseen in the active fragment of prosaposin (22-mer; SEQ ID NO: 1). Eachof these residues is underlined in Table 5.

Models of cytokine-receptor binding (Sprang and Bazan, Curr. Opin.Struct. Biol., 3:816 (1993)) have highlighted the evolutionaryconservation of a four-helical bundle structure common to manycytokines. Each of the cytokine or growth-factor sequences related tothe prosaposin-derived sequence SEQ ID NO: 1 is located between helicesA and B (AB loop) or withing helix C of the cytokine.

TABLE 5 SEQ LOCA- ID CYTOKINE SEQUENCE TION NO: ProsaposinCEFLVKEVTKLIDNNKTEKEIL —  1 hCNTF    YVKHQGLNKNINLDSVDGVP AB loop 11hIL-6       EALAENNLNLPKMAG AB loop 12 hIL-2     LQMILNGINNYKNPKLT ABloop 13 hIL-3        ILMENNLRRPNL AB loop 14 hIL1-1γ       FYLRNNQLVAGTL AB loop 15 hEPO      AEHCSLNENITVPDTKV AB loop 16hLIF    YTAQGEPFPNNVEKLCAP AB loop 17 hIL-β       FNKIEINNKLEFESA HelixC 18 hONC-M      RPNILGLRNNIYCMAQLL Helix C 19

The structurally related cytokine and growth factor-derived peptides SEQID NOS: 11 through 19 also can be useful in methods of alleviatingneuropathic pain. Peptides SEQ ID NOS: 11 through 19 can be assayed foractivity in alleviating neuropathic pain using, for example, the Chungrat model described in Example I; assays described by Wall et al., Pain7:103-113 (1979); Bennett and Xie, Pain 33:87-107 (1988); Lekan et al.,Soc. Neurosci. Abstr. 18:287 (1992) or Palacek et al., Soc. Neurosci.Abstr. 18:287 )1992), each of which is incorporated herein by reference;or other assays for neuropathic pain.

An active fragment of prosaposin or a peptide useful in alleviatingneuropathic pain can be identified by screening a large collection, orlibrary, of random peptides or peptides of interest using, for example,one of a number of animal models of neuropathic pain. Such peptides ofinterest can be, for example, the cytokine and growth factor-derivedpeptides SEQ ID NOS: 11 through 19, which have amino acid sequencesrelated to an active fragment of prosaposin (SEQ ID NO: 1). Peptides ofinterest also can be, for example, a population of peptides related inamino acid sequence to SEQ ID NO: 1 by having the conserved asparagineresidues, leucine/isoleucine residue and one or more charged residues atthe positions corresponding to the positions in which these residues arefound in SEQ ID NO: 1 but also having one or more amino acids thatdiffer from the amino acids of SEQ ID NO: 1.

Peptide libraries include, for example, tagged chemical librariescomprising peptides and peptidomimetic molecules. Peptide libraries alsocomprise those generated by phage display technology. Phage displaytechnology includes the expression of peptide molecules on the surfaceof phage as well as other methodologies by which a protein ligand is orcan be associated with the nucleic acid which encodes it. Methods forthe production of phage display libraries, including vectors and methodsof diversifying the population of peptides which are expressed, are wellknown in the art (see, for example, Smith and Scott, Methods Enzymol.217:228-257 (1993); Scott and Smith, Science 249:386-390 (1990); andHuse, WO 91/07141 and WO 91/07149, each of which is incorporated hereinby reference). These or other well known methods can be used to producea phage display library, from which the displayed peptides can becleaved and assayed for activity in alleviating neuropathic pain using,for example, one of the assays disclosed below. If desired, a populationof peptides can be assayed for activity in alleviating neuropathic pain,and an active population can be subdivided and the assay repeated inorder to isolate an active peptide from the population. Other methodsfor producing peptides useful in the invention include, for example,rational design and mutagenesis based on the amino acid sequences ofactive fragments of prosaposin such as SEQ ID NO: 1 and SEQ ID NO: 2,for example.

As disclosed herein, an active fragment of prosaposin or a peptideuseful in alleviating neuropathic pain can be identified by its activityin alleviating neuropathic pain in any of a number of well-establishedanimal models of neuropathic pain (Bennett, supra, 1993). For example,an active fragment of prosaposin can be identified using an experimentalmodel of peripheral neuropathy produced by segmental spinal nerveligation in the rat. The Chung rat model duplicates the symptoms ofhuman patients with causalgia, or burning paid due to injury of aperipheral nerve (Kim and Chung, supra, 1992). The surgical procedure ofKim and Chung produces a long-lasting hyperalgesia to noxious heat andmechanical allodynia of the affected foot. As described in Example I,rats with spinal nerve ligation according to the procedure developed byChung and Kim are useful for identifying an active fragment ofprosaposin for use in alleviating neuropathic pain.

An active fragment of prosaposin or a peptide useful in alleviatingneuropathic pain also can be identified using the neuroma model of Wallet al. This well-recognized model of neuropathic pain reproduces thehuman symptoms seen following amputation or nerve transection in anintact limb (Wall et al., supra, 1979). As discussed above, a neuromaforms readily after nerve transection due to the frustrated growth ofneurite sprouts.

A model of chronic constriction injury also can be used to identify anactive fragment of prosaposin or a peptide useful in alleviatingneuropathic pain. The chronic constriction injury model of Bennett andXie, supra, 1988, is a rat model of peripheral neuropathy that producespain disorders like those seen in man. In the Bennett model, nerveinjury is created by loosely tying constrictive ligatures around the ratsciatic nerve, causing degeneration of nerve distal to the constriction.Allodynia and hyperalgesia are produced by the constriction injury inaddition to spontaneous pain.

Primate models of neuropathic pain also are useful for identifying anactive fragment of prosaposin or a peptide useful in alleviatingneuropathic pain (see, for example, Lekan et al., supra, 1992; Palaceket al., supra, 1992).

As used herein, the term “peptide,” as used in reference to an activefragment of prosaposin, a prosaposin-derived peptide or a peptide usefulin alleviating neuropathic pain, means a compound containing naturallyoccurring amino acids, non-naturally occurring amino acids or chemicallymodified amino acids, provided that the compound retains activity inalleviating neuropathic pain. A prosaposin-derived peptide also can be apeptide mimetic, which is a non-amino acid chemical structure thatmimics the structure of a prosaposin-derived peptide and retainsactivity in alleviating neuropathic pain. Such a mimetic generally ischaracterized as exhibiting similar physical characteristics such assize, charge or hydrophobicity in the same spatial arrangement found inthe prosaposin-derived peptide counterpart. A specific example of apeptide mimetic is a compound in which the amide bond between one ormore of the amino acids is replaced, for example, by a carbon-carbonbond or other bond well known in the art (see, for example, Sawyer,Peptide Based Drug Design, ACS, Washington (1995), which is incorporatedherein by reference).

As used herein, the term “amino acid” refers to one of the twentynaturally occurring amino acids, including, unless stated otherwise,L-amino acids and D-amino acids. The term amino acid also refers tocompounds such as chemically modified amino acids including amino acidanalogs, naturally occurring amino acids that are not usuallyincorporated into proteins such as norleucine, and chemicallysynthesized compounds having properties known in the art to becharacteristic of an amino acid, provided that the compound can besubstituted within a peptide such that it retains its biologicalactivity. For example, glutamine can be an amino acid analog ofasparagine, provided that it can be substituted within an activefragment of prosaposin that retains its activity in alleviatingneuropathic pain. Other examples of amino acids and amino acids analogsare listed in Gross and Meienhofer, The Peptides: Analysis, Synthesis,Biology, Academic Press, Inc., New York (1983), which is incorporatedherein by reference. An amino acid also can be an amino acid mimetic,which is a structure that exhibits substantially the same spatialarrangement of functional groups as an amino acid but does notnecessarily have both the α-amino and α-carboxyl groups characteristicof an amino acid.

An active fragment of prosaposin or a peptide useful in the inventioncan be isolated or synthesized using methods well known in the art. Suchmethods include recombinant DNA methods and chemical synthesis methodsfor production of a peptide. Recombinant methods of producing a peptidethrough expression of a nucleic acid sequence encoding the peptide in asuitable host cell are well known in the art and are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd, Vols 1 to 3, Cold Spring Harbor Laboratory Press, New York (1989),which is incorporated herein be reference.

An active fragment of prosaposin or a peptide useful in the inventionalso can be produced by chemical synthesis, for example, by the solidphase peptide synthesis method of Merrifield et al., J. Am. Chem. Soc.85:2149 (1964), which is incorporated herein by reference. Standardsolution methods well known in the art also can be used to synthesize apeptide useful in the invention (see, for example, Bodanszky, Principlesof Peptide Synthesis, Springer-Verlag, Berlin (1984) and Bodanszky,Peptide Chemistry, Springer-Verlag, Berlin (1993), each of which isincorporated herein by reference). A newly synthesized peptide can bepurified, for example, by high performance liquid chromatography (HPLC),and can be characterized using, for example, mass spectrometry or aminoacid sequence analysis.

It is understood that limited modifications can be made to an activefragment of prosaposin without destroying its biological function. Thus,a modification of an active fragment of prosaposin that does not destroyits ability to alleviate neuropathic pain is within the definition of anactive fragment of prosaposin. A modification can include, for example,an addition, deletion, or substitution of amino acid residues; asubstitution of a compound that mimics amino acid structure or function;and addition of chemical moieties such as amino or acetyl groups. Theactivity of a modified peptide in alleviating neuropathic pain can beassayed using an animal model of neuropathic pain, such as thosedescribed above or the assay exemplified in Example I.

A particularly useful modification of an active fragment of prosaposinis one that confers, for example, increased stability. For example,incorporation of one or more D-amino acids or substitution or deletionof lysine can increase the stability of an active fragment of prosaposinby protecting against peptide degradation. For example, as disclosedherein, the prosaposin-derived 14-mer SEQ ID NO: 2 has an amino acidsequence derived from amino acids 16 to 29 of saposin C but which hasbeen modified by substitution or deletion of each of the three naturallyoccurring lysines and the addition of a C-terminal tyrosine residue. Inparticular, the prosaposin-derived 14-mer SEQ ID NO: 2 has a D-alaninefor lysine substitution at position 2; an alanine for lysinesubstitution at position 8 and a deletion of lysine at position 11. TheD-alanine substitution at position 2 confers increased stability byprotecting the peptide from endoprotease degradation, as is well knownin the art (see, for example, page 247 of Partridge, Peptide DrugDelivery to the Brain, Raven Press, New York (1991), which isincorporated herein by reference). The substitution or deletion of alysine residue confers increased resistance to trypsin-like proteases,as is well known in the art (Partridge, supra, 1991). Thesesubstitutions increase stability and, thus, bioavailability of peptideSEQ ID NO: 2, but do not affect activity in alleviating neuropathicpain.

A useful modification also can be one that promotes peptide passageacross the blood-brain barrier, such as a modification that increaseslipophilicity or decreases hydrogen bonding. For example, a tyrosineresidue added to the C-terminus of the prosaposin-derived peptide (SEQID NO: 2) increases hydrophobicity and permeability to the blood-brainbarrier (see, for example, Banks et al., Peptides 13:1289-1294 (1992,which is incorporated herein by reference, and Pardridge, supra, 1991).A chimeric peptide-pharmaceutical that has increased biologicalstability or increased permeability to the blood-brain barrier, forexample, also can be useful in the method of the invention.

One skilled in the art can readily assay the ability of an activefragment of prosaposin to cross the blood-brain barrier in vivo, forexample, as disclosed in Example II. In addition, an active fragment ofprosaposin can be tested for its ability to cross the blood-brainbarrier using an in vitro model of the blood-brain barrier based on abrain microvessel endothelial cell culture system, for example asdescribed in Bowman et al., Ann. Neurol. 14:396-402 (1983) or Takahuraet al., Adv. Pharmacol. 22:137-165 (1992), each of which is incorporatedherein by reference.

As used herein, the term “effective amount” means the amount of anactive fragment of prosaposin useful for alleviating neuropathic pain orfor preventing neuropathic pain. An effective amount to be administeredsystemically on a daily basis depends on the body weight of the subject.Preferably, an effective amount to be administered systemically on adaily basis is about 0.1 μg/kg to about 1000 μg/kg. More preferably, aneffective amount to be administered systemically on a daily basis isabout 10 μg/kg to about 100 μg/kg. An effective amount of a peptide foralleviating or preventing pain can be determined empirically usingmethods well known to those in the art, including, for example, theassay described in Example I or those disclosed above, including assaysin primates (Lekan et al., supra, 1992, and Palacek et al., supra,1992).

As used herein, the term “subject” means a vertebrate, preferably amammal and, in particular, a human.

The present invention provides a method of alleviating pain byadministering an effective amount of an active fragment of prosaposinintravenously, intramuscularly, intradermally, subcutaneously,intracranially, intracerebrospinally, topically or orally. Apharmaceutically acceptable carrier of well known type can beadministered with an active fragment of prosaposin. Such carriersinclude, for example, phosphate buffered saline (PBS).

Preferably, an effective amount of an active fragment of prosaposin isinjected directly into the bloodstream of the subject. For example,intravenous injection of an active fragment of prosaposin can be used toadminister the active fragment to the peripheral or central nervoussystem, since an iodinated prosaposin-derived 18-merTyr-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu(SEQ ID NO: 20), consisting of amino acids 12 to 29 ofprosaposin-derived 22-mer SEQ ID NO: 1 with a substitution of tyrosinefor valine at amino acid 12 (MW=2000) crossed the blood-brain barrierand entered the central nervous system as described in Example II. Theuptake by the brain was approximately 0.03%, which is in the midrange ofvalues for peptides of that approximate size that will cross theblood-brain barrier (Banks et al., supra, 1992).

Oral administration often can be desirable, provided the active fragmentof prosaposin is modified so as to be stable to gastrointestinaldegradation and readily absorbable. The substitution, for example, ofone or more D-amino acids can confer increased stability to aprosaposin-derived peptide useful in the invention.

Direct intracranial injection or injection into the cerebrospinal fluidalso can be used to introduce an effective amount of an active fragmentof prosaposin into the central nervous system of a subject. In addition,an active fragment of prosaposin can be administered to peripheralneural tissue by direct injection or local topical application or bysystemic administration. Various conventional modes of administrationalso are contemplated, including intravenous, intramuscular,intradermal, subcutaneous, intracranial, epidural, topical and oraladministration.

An active fragment of prosaposin also can be administered in a sustainedrelease form. The sustained release of an active fragment of prosaposinhas the advantage of alleviating neuropathic pain over an extendedperiod of time without the need for repeated administrations of theactive fragment.

Sustained release can be achieved, for example, with a sustained releasematerial such as a wafer, an immunobead, a micropump or other materialthat provides for controlled slow release of the active fragment ofprosaposin. Such controlled release materials are well known in the artand available from commercial sources (Alza Corp., Palo Alto Calif.;Depotech, La Jolla Calif.; see, also, Pardoll, Ann. Rev. Immunol.13:399-415 (1995), which is incorporated herein by reference). Inaddition, a bioerodible or biodegradable material that can be formulatedwith an active fragment of prosaposin, such as polylactic acid,polygalactic acid, regenerated collagen, multilamellar liposomes orother conventional depot formulations, can be implanted to slowlyrelease the active fragment of prosaposin. The use of infusion pumps,matrix entrapment system, and transdermal delivery devices also arecontemplated in the present invention.

An active fragment of prosaposin also can be advantageously enclosed inmicelles or liposomes. Liposome encapsulation technology is well known.Liposomes can be targeted to a specific tissue, such as neural tissue,through the use of receptors, ligands or antibodies capable of bindingthe targeted tissue. The preparation of these formulations is well knownin the art (see, for example, Pardridge, supra, 1991, and Radin andMetz, Meth. Enzymol. 98:613-618 (1983), which is incorporated herein byreference).

The invention also provides a method of alleviating neuropathic pain ina subject by transplanting into the subject a cell genetically modifiedto express and secrete an active fragment of prosaposin. Transplantationcan provide a continuous source of an active fragment of prosaposin and,thus, sustained alleviation of neuropathic pain. For a subject sufferingfrom prolonged or chronic neuropathic pain, such a method has theadvantage of obviating or reducing the need for repeated administrationof an active fragment of prosaposin.

Using methods well known in the art, a cell readily can be transfectedwith an expression vector containing a nucleic acid encoding an activefragment of prosaposin (Chang, Somatic Gene Therapy, CRC Press, BocaRaton (1995), which is incorporated herein by reference). Followingtransplantation into the brain, for example, the transfected cellexpresses and secretes an active fragment of prosaposin and, thus,alleviates neuropathic pain. Such a method can be useful to alleviateneuropathic pain as described for the transplantation of cells thatsecrete substances with analgesic properties (see, for example, Czechand Sagen, Prog. Neurobiol. 46:507-529 (1995), which is incorporatedherein by reference).

The cell can be any cell that can survive when transplanted and that canbe modified to express and secrete an active fragment of prosaposin. Inpractice, the cell should be immunologically compatible with thesubject. For example, a particularly useful cell is a cell isolated fromthe subject to be treated, since such a cell is immunologicallycompatible with the subject.

A cell derived from a source other than the subject to be treated alsocan be useful if protected from immune rejection using, for example,microencapsulation or immunosuppression. Useful microencapsulationmembrane materials include alginate-poly-L-lysine alginate and agarose(see, for example, Goosen, Fundamentals of Animal Cell Encapsulation andImmobilization, CRC Press, Boca Raton (1993); Tai and Sun, FASEB J.7:1061 (1993); Liu et al., Hum. Gene Ther. 4:291 (1993); and Taniguchiet al., Transplant. Proc. 24:2977 (1992), each of which is incorporatedherein by reference). For example, pain reduction has been achievedusing polymer encapsulated cells transplanted into the rat spinalsubarachnoid space (Wang et al., Soc. Neurosci. Abstr. 17:235 (1991),which is incorporated herein by reference).

For treatment of a human subject, the cell can be a human cell, althougha non-human mammalian cell also can be useful. In particular, a humanfibroblast, muscle cell, glial cell, neuronal precursor cell or neuroncan be transfected with an expression vector to express and secrete anactive fragment of prosaposin such as SEQ ID NO: 1. A primary fibroblastcan be obtained, for example, from a skin biopsy of the subject to betreated and maintained under standard tissue culture conditions. Aprimary muscle cell also can be useful for transplantation.Considerations for neural transplantation are described, for example, inChang, supra, 1995.

A cell derived from the central nervous system can be particularlyuseful for transplantation to the central nervous system since thesurvival of such a cell is enhanced within its natural environment. Aneuronal precursor cell is particularly useful in the method of theinvention since a neuronal precursor cell can be grown in culture,transfected with an expression vector and introduced into an individual,where it is integrated. The isolation of neuronal precursor cells, whichare capable of proliferating and differentiating into neurons and glialcells, is described in Renfranz et al., Cell 66:713-729 (1991), which isincorporated herein by reference.

Methods of transfecting cells ex vivo are well known in the art(Kriegler, Gene Transfer and Expression: A Laboratory Manual, W. H.Freeman & Co., New York (1990)). For the transfection of a cell thatcontinues to divide such as a fibroblast, muscle cell, glial cell orneuronal precursor cell, a retroviral vector is preferred. For thetransfection of an expression vector into a postmitotic cell such as aneuron, a replication-defective herpes simplex virus type 1 (HSV-1)vector is useful (During et al., Soc. Neurosci. Abstr. 17:140 (1991);Sable et al., Soc. Neurosci. Abstr. 17:570 (1991), each of which isincorporated herein by reference).

A nucleic acid encoding an active fragment of prosaposin can beexpressed under the control of one of a variety of promoters well knownin the art, including a constitutive promoter or inducible promoter.See, for example, Chang, supra, 1995. A particularly useful constitutivepromoter for high level expression is the Moloney murine leukemia viruslong-terminal repeat (MLV-LTR), the cytomegalovirus immediate-early(CMV-IE) or the simian virus 40 early region (SV40 ).

A nucleic acid sequence encoding an active fragment of prosaposin isdisclosed herein. For example, a nucleic acid sequence encoding SEQ IDNO: 1 is 5′-TGTGAATTCCTGGTGAAGGAGGTGACCAAGCTGATTGACAACAACAAGACTGAGAAAGAAATACTC-3′ (SEQ ID NO: 21) (Dewji et al., Proc. Natl. Acad. Sci.USA 84:8652-8656 (1987), which is incorporated herein by reference). Inorder to direct secretion of peptide SEQ ID NO: 1, for example, anucleic acid encoding a signal sequence, such as the signal sequence ofβ-lactamase, can be operably linked to SEQ ID NO: 21 as described inSimon et al., J. Cell Biol. 104:1165 (1987), which is incorporatedherein by reference.

The invention further provides a method of preventing neuropathic painin a subject by administering an effective amount of an active fragmentof prosaposin to the subject. The method of preventing neuropathic painis useful when applied prior to a painful event, for example, prior tochemotherapy or surgery that is known to result in neuropathic pain.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Alleviation Of Neuropathic Pain In Chung Model Rats

This example describes the effects of bolus intrathecal injection of anactive fragment of prosaposin in the Chung experimental model ofperipheral neuropathic pain.

Each of the three peptides were obtained in pure form by chemicalsynthesis, dissolved in sterile PBS and buffered to a neutral pH.

The surgical procedure previously described by Kim and Chung, supra,1992, was performed on male Sprague-Dawley rats weighing 120 to 150grams to induce an allodynic state. Briefly, the rats were anesthetizedwith halothane; subsequently, the left L-5 and L-6 spinal nerves wereisolated adjacent to the vertebral column and ligated with 6.0 silksuture distal to the dorsal root ganglion. After a ten to fourteen daypost operative recovery period, a spinal catheter was introduced. Fivedays following the second surgery, intrathecal drug administration wasaccomplished using a gear driven micro-injection syringe connected to aspinal catheter inserted through the foramen magnum. Prior to testing,the rats were placed in clear plastic wire meshed cages and allowed toaccommodate.

To assess the 50% mechanical threshold for paw withdrawal, a von Freyhair was applied to the hind foot avoiding the foot pad. Each of the vonFrey hairs, which are calibrated to bend at increasing log forces, werepressed perpendicularly to the foot with sufficient force to causeslight bending for a duration of approximately six to eight seconds. Apositive response was noted if the foot was sharply withdrawn. Six datapoints were collected for each point with the maximum and minimumstimulus noted for each time point. The resulting pattern of theresponses was tabulated, and the 50% response threshold was computed.The graph gives the response to the indicated dosage of peptide given asa single intrathecal bolus injection. The X-axis indicates the timeafter the injection at which point the hypersensitivity to pressure onthe foot pad was measured.

All surgically lesioned rats showed tactile allodynia prior to injectionwith an active fragment of prosaposin. As shown at time zero in FIG. 1,the measured threshold was less than 3.0 to 4.0 g in the absence ofpeptide. Intrathecal injection of 0.7 or 0.07 μg of theprosaposin-derived 22-mer peptide (SEQ ID NO: 1) suppressed allodynia ina dose-dependent fashion. The reduction of allodynia is manifest by theincrease in the force threshold as the rats withstand an increasingforce before withdrawing the affected foot.

A significant effect was observed by 15 minutes after the injection. Themaximum effect was seen 120 minutes post-injection. Rats injected withthe highest dose of the prosaposin-derived 22-mer peptide (SEQ ID NO: 1)continued to demonstrate significantly reduced allodynia at the latesttime point assayed (180 minutes). Rats that were injected with 0.007 μgprosaposin-derived 22-mer peptide (SEQ ID NO: 1) showed no significantreduction in allodynia. No significant side effects such as sedationwere observed at any concentration.

The ability of the prosaposin-derived 14-mer peptide (SEQ ID NO: 2; seeTable 1) to relieve allodynia in Chung model rats also was examined. Asshown in FIG. 2, the active fragment of prosaposin (SEQ ID NO: 2) waseffective in reducing allodynia. The peak effect of theprosaposin-derived 14-mer peptide (SEQ ID NO: 2) was observed 15 to 30minutes following the injection and returned to the pre-injection valueby 60 minutes (FIG. 2). No side effects were observed at eitherconcentration of prosaposin-derived 14-mer peptide (SEQ ID NO: 2)tested.

A mutant 22-mer peptide (SEQ ID NO: 8) that differs from theprosaposin-derived 22-mer peptide (SEQ ID NO: 1) by containing anaspartic acid residue instead of an asparagine (see Table 4) also wastested for activity in relieving allodynia in Chung model rats. Nochange in the allodynic response of the Chung rats was observedfollowing injection of 17.5 μg mutant 22-mer peptide (SEQ ID NO: 8).

Normal rats, which do not experience pain as a result of surgical lesionintroduced according to the Chung model, also were injected with anactive fragment of prosaposin (SEQ ID NO: 1) and tested for theirresponse to a heat stimulus according to the procedure developed byBennett and Xie, supra, 1988. Briefly, the period of time before the ratwithdraws the affected foot from a source of heat is defined as the hotplate latency and is a measure of tolerance to pain caused by a heatstimulus.

An intrathecal catheter was placed into normal male Sprague Dawley rats.Five days after this surgery, rats were injected intrathecally with anactive fragment of prosaposin (SEQ ID NO: 1). Rats were examined on thehot plate (52.5° C.); hot plate response latencies were measured priorto injection and at various time points up to 180 minutes after theinjection. No significant elevation of the hot plate response latencywas observed. Thus, the prosaposin-derived peptide SEQ ID NO: 1 does noteffect the perception of pain in normal animals.

EXAMPLE II In Vivo Uptake Of Prosaposin-Derived Peptide By The CentralNervous System

An 18-mer peptide (SEQ ID NO: 20) consisting of amino acids 12-29 ofsaposin C with a tyrosine substituted for valine at position 12 waschemically synthesized on an Applied Biosystems Model 430 peptidesynthesizer. The peptide was then radioiodinated by the lactoperoxidasemethod; 20×10⁶ cpm radiolabeled peptide were injected into the auriclesof rats. The animals were sacrificed after one hour and 24 hours, andthe hearts were perfused with isotonic saline in order to remove theblood from the brain.

In order to determine the percentage of peptide uptake, the brain wasthen counted in a gamma counter. In addition, the brain was homogenizedand fractionated into a capillary rich fraction (pellet) and aparenchymal brain fraction (supernatant) after dextran centrifugation(Triguero et al., J. Neurochem., 54:1882-1888 (1990), which isincorporated herein by reference). This method allows for thediscrimination between radiolabeled peptide within blood vessels andthat within the brain. After 24 hours, 0.017% of the injected peptide(SEQ ID NO: 20) was detected in whole brain; 75% of the label was in theparenchymal fraction and 25% was in the capillary fraction. At 1 hour,0.03% of the injected dose was present in whole brain.

Although the invention has been described with reference to the examplesabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

21 22 amino acids amino acid linear not provided 1 Cys Glu Phe Leu ValLys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys 1 5 10 15 Thr Glu Lys GluIle Leu 20 14 amino acids amino acid linear not provided Peptide /note=“Xaa is D-alanine” 2 Thr Xaa Leu Ile Asp Asn Asn Ala Thr Glu Glu Ile LeuTyr 1 5 10 12 amino acids amino acid linear not provided 3 Leu Ile AspAsn Asn Lys Thr Glu Lys Glu Ile Leu 1 5 10 22 amino acids amino acidlinear not provided 4 Cys Gln Phe Val Met Asn Lys Phe Ser Glu Leu IleVal Asn Asn Ala 1 5 10 15 Thr Glu Glu Leu Leu Tyr 20 21 amino acidsamino acid linear not provided 5 Cys Gln Leu Val Asn Arg Lys Leu Ser GluLeu Ile Ile Asn Asn Ala 1 5 10 15 Thr Glu Glu Leu Leu 20 22 amino acidsamino acid linear not provided 6 Cys Glu Tyr Val Val Lys Lys Val Met LeuLeu Ile Asp Asn Asn Arg 1 5 10 15 Thr Glu Glu Lys Ile Ile 20 22 aminoacids amino acid linear not provided 7 Cys Glu Phe Val Val Lys Glu ValAla Lys Leu Ile Asp Asn Asn Arg 1 5 10 15 Thr Glu Glu Glu Ile Leu 20 22amino acids amino acid linear not provided 8 Cys Glu Phe Leu Val Lys GluVal Thr Lys Leu Ile Asp Asp Asn Lys 1 5 10 15 Thr Glu Lys Glu Ile Leu 2014 amino acids amino acid linear not provided 9 Thr Lys Leu Ile Asp AsnAsp Lys Thr Glu Lys Glu Ile Leu 1 5 10 14 amino acids amino acid linearnot provided 10 Thr Lys Ser Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu1 5 10 20 amino acids amino acid linear not provided 11 Tyr Val Lys HisGln Gly Leu Asn Lys Asn Ile Asn Leu Asp Ser Val 1 5 10 15 Asp Gly ValPro 20 15 amino acids amino acid linear not provided 12 Glu Ala Leu AlaGlu Asn Asn Leu Asn Leu Pro Lys Met Ala Gly 1 5 10 15 17 amino acidsamino acid linear not provided 13 Leu Gln Met Ile Leu Asn Gly Ile AsnAsn Tyr Lys Asn Pro Lys Leu 1 5 10 15 Thr 12 amino acids amino acidlinear not provided 14 Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu 15 10 13 amino acids amino acid linear not provided 15 Phe Tyr Leu ArgAsn Asn Gln Leu Val Ala Gly Thr Leu 1 5 10 17 amino acids amino acidlinear not provided 16 Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr ValPro Asp Thr Lys 1 5 10 15 Val 18 amino acids amino acid linear notprovided 17 Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Val Glu Lys LeuCys 1 5 10 15 Ala Pro 15 amino acids amino acid linear not provided 18Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe Glu Ser Ala 1 5 10 15 18amino acids amino acid linear not provided 19 Arg Pro Asn Ile Leu GlyLeu Arg Asn Asn Ile Tyr Cys Met Ala Gln 1 5 10 15 Leu Leu 18 amino acidsamino acid linear not provided 20 Tyr Lys Glu Val Thr Lys Leu Ile AspAsn Asn Lys Thr Glu Lys Glu 1 5 10 15 Ile Leu 66 base pairs nucleic aciddouble linear not provided 21 TGTGAATTCC TGGTGAAGGA GGTGACCAAGCTGATTGACA ACAACAAGAC TGAGAAAGAA 60 ATACTC 66

What is claimed:
 1. A substantially pure polypeptide consisting of thesequence: Thr-R1-Leu-Ile-Asp-Asn-Asn-Ala-Thr-Glu-Glu-Ile-Leu-Tyr;wherein R1 is D-alanine.
 2. A composition comprising the polypeptide ofclaim 1 and a pharmaceutically acceptable carrier.